Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: Cell death induction by 1H13-survivin/BIRC5-derived peptides targeted to the cytoplasm, mitochondria, or nucleus (A) The sequences added to the 1H13-BIRC5 peptide to target it to the: (1) cytoplasm, (2) mitochondria, or (3) nucleus. The underlined sequence represents the 1H13 peptide; in red, amino acids indicate D-amino acid substitutions. (B) A549 cells were incubated for 90 min with 5 μM FITC-labeled, nucleus-targeted peptide IH13-Nuc, and were also IF-stained with anti--SMAC, anti-IP3R or anti-GM130 antibodies, and stained with DAPI to visualize the mitochondria, ER, Golgi, and nucleus, respectively. Confocal microscope images are shown, with white arrows indicating peptide co-localization with the mitochondria (SMAC). Orange and yellow arrows indicate peptide presence in the nucleus and cytosol, respectively. (C) A549 cells were incubated with the mitochondria- or nucleus-targeted 1H13-BIRC5-derived peptide for 24 h in serum-free medium, followed by a cell proliferation assay using the SRB method. (D and E) Apoptotic cell death as induced in A549 cells following incubation for 24 h with the nucleus-targeted peptide (2/3D-1H13-Nuc) in the presence or absence of the indicated concentrations of the peptides in serum-free medium and subjected to FITC–annexin V/PI staining, followed by a flow cytometry analysis. Representative histograms for control and selected peptide concentration (D) and analysis of early and late apoptotic stages are shown (E). (F and G) Cell death as induced by 2/3D-1H13-Nuc in different cell lines, A549, SH-SY5Y, U-87MG, PC-3, and HUV-EC-C (F) or Jurkat, K562, and KMH2-LC (G) were treated with the indicated concentrations of the peptide for 24 h, then subjected to cell death analysis using propidium iodide (PI) staining and flow cytometry. (H and I) A549 cells were seeded at a density of 2 × 10 5 cells per well in a 12-well plate. After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions. Twenty-four hours post-transfection, the cells were re-seeded at 1 × 10 5 cells per well in a 12-well plate. After another 24 h, the culture medium was replaced with serum-free medium, and the cells were treated with the indicated concentrations of the 2/3D-1H13-Nuc peptide. Survivin overexpression levels were assessed by immunoblotting (H), and cell death was analyzed by propidium iodide (PI) staining followed by FACS analysis (I). Results represent the means ± SEM ( n = 3).

Article Snippet: After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions.

Techniques: Derivative Assay, Sequencing, Incubation, Labeling, Staining, Microscopy, Proliferation Assay, Flow Cytometry, Control, Concentration Assay, Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: Schematic drawing of the multimerization modes of DNMT3 complexes and of the dinucleosome substrates used in this study. A , protein/protein interaction interfaces present on DNMT3A, DNMT3B, DNMT3L, and DNMT3B3 subunits. The RD interface also provides the DNA binding site. B , protein multimerization of different DNMT3 complexes. DNMT3A and DNMT3B can form large homomultimers; homotetramers with one central RD interface are the smallest catalytically active species. DNMT3A/3L and DNMT3A/3B3 form defined heterotetramers. C , multimerization of DNMT3 complexes binding next to each other on DNA. D , dinucleosomes used as methylation substrates in this study. The CpG sites in the linker DNA region are highlighted and annotated. The regions used for bisulfite sequencing of the top and bottom DNA strand are indicated. The parts of the 70 bp linker removed in the 58(1) and 58(2) linkers are indicated. The NNN part between CpG 5 and CpG 6 denotes an internal bar code, used to discriminate the NGS data from pooled substrates. NNN = TGT in Linker-70, TCA in Linker-58(1), CTA in Linker-58(2). NGS, next generation sequencing.

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Binding Assay, Methylation, Methylation Sequencing, Next-Generation Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: Linker methylation patterns of all CpG sites in the three dinucleosomes by DNMT3AC/3B3C. A , example of top strand and bottom strand methylation data. Values were normalized to the average methylation of linker CpG sites. B , relative linker methylation levels observed in five independent experiments. In panel A and B , no methylation data were indicated for the missing CpG sites 1 to 3 in Linker-58(1), and 6 to 8 in Linker-58(2). C , difference in the relative methylation levels of equivalent CpG sites between Linker-70 and Linker-58(1) or 58(2). The regions missing in the Linker-58 nucleosomes are shaded in gray . Error bars represent the propagated standard deviations. D , p values for the significance of the methylation differences shown in panel C determined by two-sided t test with equal variance using the individual data points. Significant p values ( p < 0.05/26, considering multiple testing) are shaded in gray .

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Methylation

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: DNMT3AC/3B3C methylation of linker CpG sites determined by competitive methylation of all three dinucleosome substrates. A , average linker methylation levels of all three dinucleosomes, methylated in competition in one reaction tube. Exemplary methylation profiles are shown in . The table provides p values for the pairwise comparison of the methylation levels determined by two-sided t test with equal variance. Significant p values ( p < 0.05/3, considering multiple testing) are shaded in gray . B , enrichment of sequence reads with many methylation events in the entire data set. The distribution of the total number of methylation events per sequence read was determined. Based on the average methylation level of the pool, an expected number of sequence reads with each number of methyl groups was calculated by binomial statistics and the observed/expected ratio (obs/exp) determined.

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Methylation, Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: Results of the competitive methylation of dinucleosomes linker regions and free DNA by DNMT3AC, DNMT3AC/3B3C, and DNMT3AC/3B3C-RE mutant. A , relative CpG site methylation levels of free DNA determined in four independent competitive methylation experiments. The sequence and CpG site annotation of the free DNA is provided in A . The 6 most preferred sites based on DNMT3A flanking sequence preferences literature data are shaded in gray . B , relative methylation levels of linker DNA in the Linker-70 dinucleosomes substrate determined in the four independent competitive methylation experiments. C , heatmap comparing the relative DNMT3AC/3B3C activity (rel. Act.) on free DNA and Linker-70 linker DNA with the flanking sequence preferences of the corresponding CpG sites (Pref) sorted by the average of both columns after scaling. Pearson r-values are indicated below. The scatter plots showing the correlations of the heatmaps are provided in , B and C . D , ratio of the average methylation activities of DNMT3AC, DNMT3AC/3B3C, and DNMT3AC/3B3C-RE on dinucleosomes and free DNA. The table provides the p values of pairwise comparisons based on two-sided t test with equal variance. Significant p values ( p < 0.05/3, considering multiple testing) are shaded in gray .

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Methylation, Mutagenesis, Sequencing, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: Effects of the recruitment of DNMT3AC/3B3C to the nucleosome. A , comparison of the flanking sequence preferences of DNMT3A and the relative DNMT3AC/3B3C activity at all linker CpG sites. The error bars show the standard deviation. Individual data points are provided in . Thirteen out of the 26 sites with significant difference and deviations > 10% are shaded gray . p -values were determined by Z-statistics and p < 0.05/26 (considering multiple testing) was indicated as significant. B , Visualization of the sites showing the strongest nucleosomal effects on methylation rates on a dinucleosome model generated by rigid body superposition as described in the Methods section. Top strand CpG (in the notation of this study) are colored red , bottom strand CpG orange . The position of sites with most prominent nucleosome dependent stimulation or repression of activity are highlighted by green (increased activity) or red arrows (reduced activity). The two images show the same model rotated by approximately 90° about the linker DNA axis.

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Comparison, Sequencing, Activity Assay, Standard Deviation, Methylation, Generated

Journal: The Journal of Biological Chemistry

Article Title: Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3 complexes on DNA

doi: 10.1016/j.jbc.2026.111154

Figure Lengend Snippet: Schematic picture of the multimerization of DNMT3AC/3B3C complex heterotetramers on dinucleosomes. CpG cytosine residues that can easily reach a DNMT3A active site after base flipping are methylated more efficiently than expected by their flanking sequence (symbolized by gray shading), CpG cytosine residues placed such that they cannot bind to an active site are methylated only weakly (symbolized by the white color). The DNMT3 complexes and nucleosome are not drawn to scale.

Article Snippet: For the methylation experiments with different dinucleosome substrates, each dinucleosome variant was digested with MluI-HF (NEB) for 15 min at 37 °C in 7 μl NEB Cutsmart buffer (50 mM KOAc/20 mM Tris-acetate pH 7.9, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin).

Techniques: Methylation, Sequencing

Journal: Human Genetics and Genomics Advances

Article Title: Asparaginyl-tRNA synthetase ( NARS1 ) variants implicated in dominant neurological phenotypes display dominant-negative properties

doi: 10.1016/j.xhgg.2025.100519

Figure Lengend Snippet: Localization and conservation of NARS1 variants (A) NARS1 functional domains are indicated in gray (unique N-terminal extension), beige (anticodon binding domain), and blue (catalytic domain). The position of each NARS1 variant is displayed across the top. (B) Phenotypes associated with each NARS1 variant are provided. Green indicates a PNS-only phenotype while blue indicates PNS and CNS features. (C) The conservation of affected amino acid residues. The position of each variant is indicated alongside neighboring NARS1 amino acid residues from evolutionarily diverse species. The position of the affected residue is indicated by bold, red text. Green shading indicates a PNS-only phenotype while blue shading indicates PNS and CNS features.

Article Snippet: To evaluate the function of NARS1 variants, a p413 vector (ATCC, no. 87370) with no NARS1 insert (“Empty”) was transformed into the ptetO7- DED81 strain.

Techniques: Functional Assay, Binding Assay, Variant Assay, Residue

Journal: Human Genetics and Genomics Advances

Article Title: Asparaginyl-tRNA synthetase ( NARS1 ) variants implicated in dominant neurological phenotypes display dominant-negative properties

doi: 10.1016/j.xhgg.2025.100519

Figure Lengend Snippet: NARS1 variants do not ablate protein expression or dimerization (A) Yeast protein lysates were subjected to western blot assays to detect human NARS1 proteins expressed from wild-type and mutant constructs, as indicated along the top. (B) Quantification of NARS1 protein expression. Green corresponds to variants associated with peripheral neuropathy and blue corresponds to variants associated with a peripheral and central nervous system phenotype. One-way ANOVA with Dunnett’s multiple comparison testing was used to determine statistical significance. (C) NanoLuc luciferase activity was expressed relative to firefly luciferase activity and then normalized to wild-type/wild-type activity. HEK293T cells expressing wild-type NARS1 C-LgBiT and HaloTag-SmBiT, as well as wild-type NARS1 C-LgBiT and wild-type NARS1 N-SmBiT independently, were used as controls. The normalized ratio is depicted on the y axis. Green corresponds to variants associated with peripheral neuropathy and blue corresponds to variants associated with a peripheral and central nervous system phenotype. One-way ANOVA with Dunnett’s multiple comparison testing was used to determine statistical significance. ∗ p = 0.015, ∗∗∗∗ p < 0.0001.

Article Snippet: To evaluate the function of NARS1 variants, a p413 vector (ATCC, no. 87370) with no NARS1 insert (“Empty”) was transformed into the ptetO7- DED81 strain.

Techniques: Expressing, Western Blot, Mutagenesis, Construct, Comparison, Luciferase, Activity Assay

Journal: Human Genetics and Genomics Advances

Article Title: Asparaginyl-tRNA synthetase ( NARS1 ) variants implicated in dominant neurological phenotypes display dominant-negative properties

doi: 10.1016/j.xhgg.2025.100519

Figure Lengend Snippet: NARS1 variants are associated with reduced yeast cell growth (A) Yeast containing a doxycycline-repressible element upstream of the endogenous DED81 locus (the yeast ortholog of NARS1 ) were co-transformed with an empty, low-copy p413 vector and either wild-type or mutant NARS1 in a high-copy pAG425 vector. (B) Resulting cultures were plated on galactose and raffinose media lacking leucine and histidine, and including 10 μg/mL of doxycycline. NARS1 variants are indicated across the top and serial dilutions are noted on the left. Images were taken after 3 days of growth (top) and after 5 days of growth (bottom).

Article Snippet: To evaluate the function of NARS1 variants, a p413 vector (ATCC, no. 87370) with no NARS1 insert (“Empty”) was transformed into the ptetO7- DED81 strain.

Techniques: Transformation Assay, Plasmid Preparation, Mutagenesis

Journal: Human Genetics and Genomics Advances

Article Title: Asparaginyl-tRNA synthetase ( NARS1 ) variants implicated in dominant neurological phenotypes display dominant-negative properties

doi: 10.1016/j.xhgg.2025.100519

Figure Lengend Snippet: Pathogenic NARS1 variants cause dominant growth defects in yeast when co-expressed with wild-type NARS1 (A) Yeast containing a doxycycline-repressible element upstream of DED81 (the yeast ortholog of NARS1 ) were co-transformed with wild-type NARS1 in the p413 vector and either wild-type or mutant NARS1 in the pAG425 vector. (B) Resulting cultures were plated on galactose and raffinose media lacking leucine and histidine, with 10 μg/mL of doxycycline. NARS1 variants are indicated across the top and serial dilutions are noted on the left. Images were taken after 3 days of growth (top) and after 5 days of growth (bottom). (C) Yeast spot intensity at the 1:100 dilution was measured using ImageJ analysis and then normalized to the “WT + Null” sample. Error bars indicate the standard deviation. To test for statistically significant differences in yeast growth between each strain and the WT + Null strain, a one-way ANOVA with Dunnett’s multiple comparisons test was used. ∗∗ p = 0.002, ∗∗∗∗ p < 0.001. (D) Normalized mean gray density as described in (C). To test for statistically significant differences among all strains, a one-way ANOVA with Tukey’s multiple comparisons test was used. Given that G509S does not exert a dominant-negative effect, samples were not compared with this allele (indicated by a striped blue bar). ∗ p = 0.01, ∗∗ p = 0.007, ∗∗∗ p = 0.0001.

Article Snippet: To evaluate the function of NARS1 variants, a p413 vector (ATCC, no. 87370) with no NARS1 insert (“Empty”) was transformed into the ptetO7- DED81 strain.

Techniques: Transformation Assay, Plasmid Preparation, Mutagenesis, Standard Deviation, Dominant Negative Mutation

Journal: Human Genetics and Genomics Advances

Article Title: Asparaginyl-tRNA synthetase ( NARS1 ) variants implicated in dominant neurological phenotypes display dominant-negative properties

doi: 10.1016/j.xhgg.2025.100519

Figure Lengend Snippet: DED81 rescues the dominant growth phenotype caused by pathogenic NARS1 variants (A) Yeast containing a doxycycline-repressible element upstream of DED81 (the yeast ortholog of NARS1 ) were co-transformed with wild-type NARS1 in the p413 vector and either wild-type or mutant NARS1 in the pAG425 vector. (B) Resulting cultures were plated on galactose and raffinose media lacking leucine and histidine, and with no doxycycline. NARS1 variants are indicated along the top and serial dilutions are noted on the left. Images were taken after 3 days of growth (top) and after 5 days of growth (bottom). (C) Yeast spot intensity at the 1:100 dilution was measured using ImageJ analysis and then normalized to the “WT + Null” sample. Error bars indicate the standard deviation. To test for statistically significant differences in yeast growth between each strain and the WT + Null strain, a one-way ANOVA with Dunnett’s multiple comparisons test was used. ∗ p = 0.0356, ∗∗ p = 0.0018.

Article Snippet: To evaluate the function of NARS1 variants, a p413 vector (ATCC, no. 87370) with no NARS1 insert (“Empty”) was transformed into the ptetO7- DED81 strain.

Techniques: Transformation Assay, Plasmid Preparation, Mutagenesis, Standard Deviation

Journal: Human Genetics and Genomics Advances

Article Title: Asparaginyl-tRNA synthetase ( NARS1 ) variants implicated in dominant neurological phenotypes display dominant-negative properties

doi: 10.1016/j.xhgg.2025.100519

Figure Lengend Snippet: NARS1 variants retain dominant growth defects when expressed from a low-copy number vector (A) Yeast containing a doxycycline-repressible element upstream of DED81 (the yeast ortholog of NARS1 ) harboring wild-type NARS1 in the p413 vector were co-transformed with either wild-type or mutant NARS1 in the pAG415 vector. (B) Resulting cultures were plated on galactose and raffinose media lacking leucine and histidine, and containing 10 μg/mL of doxycycline. NARS1 variants are indicated along the top and serial dilutions are noted on the left. Images were taken after 3 days of growth. (C) Yeast spot intensity at the 1:100 dilution was measured using ImageJ analysis and then normalized to the “WT + Null” sample. Error bars indicate the standard deviation. To test for statistically significant differences in yeast growth between each strain and the WT + Null strain, a one-way ANOVA with Dunnett’s multiple comparisons test was used. ∗∗∗∗ p < 0.001.

Article Snippet: To evaluate the function of NARS1 variants, a p413 vector (ATCC, no. 87370) with no NARS1 insert (“Empty”) was transformed into the ptetO7- DED81 strain.

Techniques: Low Copy Number, Plasmid Preparation, Transformation Assay, Mutagenesis, Standard Deviation